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284

OBSERVATIONS ON THE FORCES OF MORPHOGENESISIN THE AMPHIBIAN EMBRYO

BY C. H. WADDINGTON

Zoological Department and Strangeways Research Laboratory, Cambridge

(Received 15 October 1942)

(With Two Text-figures)

INTRODUCTION

Recent years have seen considerable advances in our knowledge of chemical interactionsbetween different parts of developing embryos, and of the metabolic processes by whichthe stimulating evocators are released. We have also acquired further information, on thebiological level, of the correlations between parts which lead to the formation of unitsorganized into definite patterns. On the other hand, the forces which actually bringabout- the changes in shape which are perhaps the salient feature of early developmenthave remained almost unstudied. Several hypotheses have been put forward. Thus someyears ago, Glaser (1914, 1916) suggested that the folding of the neural plate into a grooveis due to a weakening of the inner surface of the neural epidermis followed by an imbibi-tion of water; the swelling consequent on this is supposed to extend to weakened innersurface more than the stronger outer one, so that the cells are deformed into truncatedcones, and the whole layer folds. This conception has recently been criticized by Brown,Hamburger & Schmitt (1941) on the basis of accurate density determinations, which failedto reveal any evidence of imbibition. They suggest that the shape changes are primarilydue to 'an increase in the "attractive" forces between molecules in the adjoining cellsurfaces of prospective neural tissue cells so that the area of contact is actively increased'.Without making any hypotheses as to the physico-chemical mechanisms involved, Holt-freter(i939) has also emphasized the importance of attractive or repulsive forces dependenton the surface properties of the cells; in his case he was concerned with interactionsbetween masses of tissue differing from one another in histological type. Finally, severalauthors (e.g. Harrison, 1936; Needham, 1936; Waddington, 1940) have drawn attentionto the possibility that the facts could be explained if we could postulate the formation ofsubmicroscopic fibrils (an orientated cyto-skeleton) within the cytoplasm.

The present communication presents data which bear on these possibilities from anumber of angles. It had been hoped to record the observations in a more elaborate andstatistical form, but as it seems improbable that the work can be continued in the im-mediate future, it has seemed best to make an interim report on the material, which, asfar as it goes, is fairly straightforward and unlikely to be substantially altered by morequantitative study.

1. THE ORIENTATION OF YOLK GRANULES

At first sight the most obvious way of testing the suggestion that there is an orientatedcyto-skeleton would be by the use of polarized light. Observations of this kind have beenmade on the chick embryo by Hobson (1941); and there are of course many studies onthe eggs of invertebrates, where, however, the anisotropy has not in general been corre-

The forces of morphogenesis in the amphibian embryo 285

lated with form changes. In the amphibian embryo, this simple method cannot be satis-factorily used owing to the presence of large numbers of highly refractive yolk granules.These effectively obscure any double refraction which might be shown by the cytoplasm.The only recorded exception to this is in the long flask-shaped cells lining the earlyblastopore lip, where Waddington & Picken (cf. Waddington, 1940) observed a weakdouble refraction in the terminal processes, which are free of yolk, probably because theyare too thin to contain the granules.

The presence of yolk granules, although it prevents the use of polarized light, canitself be made the basis of a method of studying a possible cyto-skeleton. The granules inmost species of Amphibia are not perfect spheres, but are somewhat elongated ellipsoids.If there is a strongly orientated fibrillar structure in the cytoplasm, it would be expectedthat the granules would also be orientated with their long axes parallel to the fibrillardirection. This possibility has been investigated in a number of situations in which itmight be expected to occur in embryos of Triton alpestris and taeniatus.

(a) In the mitotic spindle

It is becoming generally accepted that the mitotic spindle is a tactoid in which fibrousprotein molecules are orientated in parallel (reviews by Darlington, 1939; Waddington,1939a). The conditions are therefore such as should lead to the orientation of yolkgranules. The spindle is, however, normally empty of granules, being formed of clearcytoplasm partly derived from the nucleus. Only rare cases of included granules havebeen found. In all these the orientation of the granules has been very exact, the long axisbeing parallel to that of the spindle. There seems no doubt therefore that yolk granulesdo become orientated if they lie in a region of cytoplasm which has a strongly fibrillarstructure.

(b) In the immediate neighbourhood of an elongating spindle

The «ietaphase spindle in amphibian embryonic cells is rather short,and broad.During anaphase it elongates considerably, pushing out in two directions through theyolky cytoplasm. If there was any pre-existing orientated skeleton in this cytoplasm, orif the cytoplasm was highly viscous, one would expect the elongating spindle to cause an.'orientation of the yolk granules in its immediate neighbourhood. This does not seem tooccur. At metaphase the ends of the chromosomes protrude in a haphazard way into thecytoplasm, lying between and amongst the yolk granules. In spite of this, no evidenceof orientation is visible in the granules immediately against the sides of anaphase spindles.

(c) In the flask cells of the young blastopore

In the two situations just mentioned, orientation might have been expected within acell, but this would not necessarily have had any particular relation to morphogeneticdevelopments. In this and the next two sections, evidence will be presented as to theorientation in cells whose shape is definitely connected with morphogenesis.

As has already been mentioned, the cells lining the early blastopore in the younggastrula become drawn out into long 'flask-shaped' ovals, with a thin neck reachingdown to the blastopore from the thicker body which lies farther within the embryo. Inthe thinnest ends of the necks, no yolk granules are present, and the material has beenshown to be anisotropic. In sections, however, the yolk granules within the body of thecells are found not to be orientated to a noticeable extent (Fig. 1 a). The granules which

286 C. H. WADDINGTON

lie farthest down in the neck may, indeed, have their long axes parallel to that of the cellbut this seems only to be true when they are so narrowly confined that there is no roomfor them to lie in any other direction. Farther inwards, in the wider regions of the cell,the granules seem to lie completely at random. A full statistical analysis would be necessaryto establish this strictly, but at least the appearances make it clear that any orientationwhich occurs is very much less than could account for the elongation of a more or lessspherical cell into the long thin flask shape.

Fig. i. a, Camera lucida drawing of the terminal ends of some 'flask-cells' extending down to the earlyblastoporal groove at the bottom right. Yolk granules indicated by hollow ovals, nuclei lie further towardsthe centre of the egg and are not visible, b and c, Similar drawings of groups of cells from the just closedneural tube; nuclei hatched, d, Lens vesicle shortly after becoming free from the comeal epithelium.

(d) In the neural groove

The changes of shape in the cells forming the neural plate, groove and tube have notyet been adequately described. Many authors have drawn attention to the elongation ofthe originally cuboidal cells of the ectodermal epithelium into a columnar shape, whichthen narrows on the outer surface to form a thin truncated cone. These changes do not,however, take place simultaneously over the whole area of the neural plate. In particular,the first change from a column towards a cone, which initiates the folding of the plate,occurs towards the edges of the neural area. The externally visible effect correlated with


pis is the elevation of the neural ridges, outlining the still unfolded neural plate. Itappears probable that a detailed study of the cell changes proceeding during these earlystages in the folding would limit the types of hypothesis which could be advanced toaccount for the phenomena.

At present, however, we are concerned only with the orientation of yolk granuleswithin the elongated columnar or conical cells. The granules in these cells appear to bein process of fairly rapid utilization, and they are not always easy to distinguish; thewell-formed granules are accompanied by masses or more or less amorphous yolkymaterial which is partially digested. The granules whose shape remains definite havebeen drawn in the figures, the other material being omitted (Fig. 1 b, c).

It is apparent that there is no noticeable orientation even in the very elongated cellsof the early closed neural tube. In these cells the nucleus is usually long and oval in shape.Its long axis always lies parallel to that of the cell as a whole. It might be suggested thatthis is evidence that the cytoplasm in which the nucleus lies has an orientated structure.It is clear, however, that the cells are often so narrow that the nucleus couid not be fittedin in any other way. The orientation of the nucleus can probably be more plausiblyconsidered as a direct consequence of constriction due to the narrowing of the cell.

(e) In the lens

During the first stage of the formation of the lens in Triton the cells of the inner layerof the ectoderm elongate, forming a single-layered columnar epithelium. This th^n foldsinwards, and becomes cut off as a small vesicle. Both in the columnar and the vesiclestages, the individual cells are considerably longer and narrower than in the originalepithelium. No trace of orientation of yolk granules can be found (Fig. id), except forsuch as are confined in very narrow spaces between cell surfaces; in such cases theorientation can be attributed to constriction by these surfaces rather than to a cytoplasmicmicro-structure.

By the time the lens fibres are formed, the yolk granules have disappeared.

2. THE SHAPE OF CELLS IN MOVING CELL STREAMS

The investigation of early development by means of marks made with vital dyes hasrevealed the frequent occurrence of considerable translocations of cells by means ofstreaming movements. Very little is known about the physical forces involved in thesemovements, although preliminary measurements of their order of magnitude have beenmade by Waddington (19396). Hypotheses as to the nature of the forces have to takeaccount of the fact, recorded by many authors (e.g. Spemann, 1931; Waddington, 1942),that small packets of cells, transplanted into other regions, retain specific capacities tomove in definite directions. This argues for the existence of some fixed polarity in thecells.

A conceivable basis for such a polarity would be a fibrillar organization of the cyto-plasm, with orientation of the fibrils. A cyto-skeleton of this kind might have visibleeffects either on the orientation of the yolk granules or of the cell shape. Fixed andstained preparations of streaming regions of amphibian embryos have therefore beenexamined in an attempt to detect such effects; the material used was the roof of theprimitive gut of a mid-gastrula and the overlying presumptive neural tissue (Fig. za, b).No evidence of any orientation of yolk granules could be detected. Moreover, as the


figures show, the cells are sensibly iso-dimensional, with no marked elongation in t h |direction of movement. Even the mitoses are not regularly orientated, and this is truenot only in the tissue some distance away from the lip of the blastopore, but in the cellsin its immediate neighbourhood, where the movement is most vigorous.

3. THE TENSILE STRENGTH OF THE CELL SURFACES

It is clear that alterations in the surface forces of the cells of different regions of theembryo, or of different parts of single cells, might produce changes in the shape oftissues or organ rudiments. No systematic attempts to detect such alterations seem tohave been made. Almost our only source of information as to the surface forces in em-bryonic amphibian cells is the investigation of Harvey & Fankhauser (1933). Theymeasured the tension at the surface of the newly fertilized egg of Triturus (Diemyctylus)viridescens by observing the flattening of the egg under the influence of gravity. The value

Fig. 2. a, A group of cells from the roof of the primitive gut of a mid-gastrula; they are moving in thedirection of the top of the page, b, A group of cells of the presumptive neural plate overlying the last; theoutline of the intercepts of the cells with the surface of the gastrula is indicated; the cells are moving towardsthe bottom of the page.

obtained was extremely low, less than 1 dyne/cm. Similarly, low values are typical of'naked protoplasm5 in general (cf. Newton Harvey, 1937). It cannot, however, beassumed that the surfaces of embryonic cells can always be considered to fall under theheading of 'naked protoplasm'. For instance, the newly fertilized eggs were studied byHarvey & Fankhauser immediately after they had secreted from the surface a fertilization(or vitelline) membrane whose strength is of quite a different order of magnitude. Thereis no reason why the cells should not in later development produce similar surfaces,whose strength might thus be considerably greater than that measured on the strippedegg-

Casual observations made during the normal operations of experimental embryologysuggest that something of the kind may be true. Small fragments of tissue being trans-ferred from place to place in pipettes sometimes come in contact with the water-airinterface. Fragments of young embryos are immediately torn to pieces by the surfaceforces, but the tissues of much older embryos (late tail-buds and swimming stages) are


pbviously tougher, and may be quite uninjured. This suggests a possible method ofinvestigating the tensile strength of the cell surfaces.

Two methods have been used for testing the reactions of cells to applied surface forces.In the first, fragments of tissue were lifted by needles into the air-liquid interface ofsolutions of saponin, whose surface tension had been measured. In the second, thetissues were brought into the interface between water and various organic liquids. Theembryos used are T. taeniatus.

After preliminary tests of a number of different sagonin concentrations, solutions intap water were made up of the dilutions 2 x io~5, io~8 and 5 x icr6 by weight. Thesurface tensions of these were measured by pulling a clean platinum wire through thesurface. The values obtained (three measurements on each solution) were: solution 1,50-4 dynes/cm.; solution 2, 55-8 dynes/cm.; solution 3, 6o-6 dynes/cm. The tap water,measured at the same time, gave a value of 73-6 dynes/cm.

At the surface of all three solutions, naked newly fertilized eggs broke immediatelyand completely, as might be expected. In unfertilized eggs which had been kept atroom temperature for 3 days, the surface had become extremely solid, and was notdisrupted at the surface of any of the solutions or even of tap water.

Tissues of embryos ranging from young gastrulae to.early tail-buds were studied.Tissues from all regions, in all stages, were broken on the surface of solution 3, with thehighest surface tension. Differential behaviour was, however, shown with respect tothe other two solutions with lower tensions. The ectoderm of the mid-gastrula wasbroken, though rather slowly, on solution 1, and rapidly on solution 2. By the earlyneural plate stage, the neural plate itself survived on solution 1, though the epidermisbroke slowly. In the neural groove stage and later, both neural and epidermal tissuessurvived on this solution. Beginning in the open neural plate.stage, a most interestingdifferential reaction of the neural tissue to solution 2 began to be manifested. If trans-verse sections of the neural tissue were carefully lifted into the surface, it was apparentthat the inner surface of the neural tissue broke more readily than the outer. The differ-ence was particularly marked in the neural groove stage, at which the inner convexsurface broke down slowly while the outer concave surface survived uninjured. By theearly tail-bud stage, neither surface of the neural tube was broken on solution 2, althoughthe tension was sufficient to tear open the just-closing neural ridges, and flatten the tubeinto a shallow groove.

The epidermis was throughout slightly weaker than the neural tissue. The mesodermwas weaker still, but survived on solution 1 by the early tail-bud stage. The endodermwas the weakest tissue of all, failing on solution 1 even in the tail-bud stage. Theseobservations are summarized in Table 1.

In using the second method, the interfacial tensions between tap water and thevarious liquids was not directly measured, but reference was made to physical tables(Landolt-Bornstein, 1912) where values of sufficient accuracy could be obtained. It wasclear that the tissues used in these experiments were very rapidly injured by the quan-tities of the substances which dissolved in the water, and all tests were therefore made asquickly as possible; even so, probably no great reliance should be placed upon them,except as confirming the general picture emerging from the experiments with saponinsolutions.

In general, tissues raised into the water-organic solvent interface broke at lower surface


tensions than those recorded for the saponin solutions. Thus all tissues tested broke ofthe water-carbon tetrachloride interface, with a tension of about 45 dynes/cm., althoughthe neural tube of tail-bud embryos was only slowly disrupted. This neural tissue sur-vived on the water-benzene interface, with a tension of about 35 dynes/cm., but othertissues (epidermis and endoderm) broke. Rather surprisingly, the epidermis was brokeneven on the water-chloroform interface (about 25 dynes/cm.), but this had no effect onthe neural tissue or notochord of mid-tail-bud stages. The epidermis survived the lowest

Table 1. Behaviour of tissues in solutions 1 (surface tension 50-4 dynes/cm.)and 2 (55-8 dynes/cm.)

Unfertilized egg freshMid-gastrula:

EctodermMesoderm

EndodermEarly neural plate:

Neural plateMesodermEndoderm

Neural groove:Neural tissue

Epidermis

Early tail bud:Neural tube

Epidermis

Mesoderm

Solution 1

Broke immediately

Broke slowlyBroke, surface of primitive

gut going slowlyBroke quickly

SurvivedBroke slowlyBroke quickly

Survived

Survived

Survived

Survived

Survived

Solution 2

—

Broke quicklyBroke quickly

Broke immediately

Lower surface broke morequickly than upper

Difference between surfacesvery marked

Torn off mesoderm, butbroke only slowly

Torn open but cells notdisrupted

Survived, but cut edgestorn away from mesoderm

Broke slowly

tension applied, that of the water-ether interface (about 11 dynes/cm.). At the tail-budstage, the endoderm was the only tissue which could not support this tension, and itwas only slowly disrupted. In the earlier stages, however (open neural plate and younger),all tissues were broken on this interface.

DISCUSSION

In the first two parts of thi9 paper, phenomena were examined which might have beenexpected to reveal evidence of a fibrillar micro-structure of the cytoplasm. No suchevidence appeared. The fact that yolk granules can be orientated in the mitotic spindleshows that such an orientation is possible in fibrillar surroundings. Its absence in thecytoplasm of cells undergoing changes in shape is therefore probably significant. Atleast it may be concluded that if any fibrillar cyto-skeleton is present, the forces exertedby the fibrils are much smaller than those in the spindle tactoid. It then becomesdifficult to suppose that they can be strong enough to play any important part in bringingabout the shape changes of morphogenesis.

The third part of the paper describes changes in surface phenomena which may be ofmore importance in this connexion. It should be said at the outset that the quantitativedata are of an extremely rough nature. The surface of saponin solutions is known to bepeculiar in many respects (Gaddum, 1932), since its tension depends on the length of


time it has been in existence and the rapidity with which it is stretched. The values forthe surface tension given in the table were measured by a method which is not directlycomparable with the phenomena which may be expected to occur when material issuddenly introduced into the surface, and therefore do not accurately represent theforces to which the embryonic cells were subjected. Moreover, it is possible that thesaponin solutions had noticeable effects on the surfaces of the cells immersed in them;and this was certainly the case with the organic solvents used.

It can hardly be supposed, however, that the uncertainties in the measurements ofthe surface tensions of the saponin solutions affected the order of magnitude estimated,or the relative tensions df the different dilutions. The order of magnitude was almost ahundred times greater than the tensions measured in the naked surfaces of similar eggsby Harvey & Fankhauser. The greater strength of the cells of the later embryonic stagescannot be attributed to the effects of the saponin or the organic solvents, since thesewould be expected to diminish surface strength rather than increase it. It therefore seemsjustifiable to draw, from the present experiments, the first conclusion that the surfacestrength of cells in the gastrula and later stages is of an altogether higher order than thatof 'naked protoplasm', being in the region of some tens of dynes per centimetre.

The second conclusion which seems justifiable is perhaps even more important. It isthat the surface strength is not the same in all cells at all times. The evidence con*sistently indicates a gradual increase in strength throughout the gastrula to tail-budstages. This affects all tissues, but not all equally. During these stages, the ectodermaltissues are always the strongest and the endodermal the weakest (in later stages thenotochord seems rapidly to acquire considerable strength). Of the ectodermal tissues,the neural material is the strongest, and it shows behaviour which is perhaps the mostinteresting of all. The fact that the upper, concave, surface of the neural groove is strongerthan the lower, convex, surface is exactly what would be expected if the surface tensionsof the cells were responsible for the folding of the neural plate into the neural tube.

Although the evidence is thus in agreement with such a hypothesis, it cannot by anymeans be taken to prove it. In the first place, what has been measured is the passivestrength of the cell surfaces against breakage by an applied force. In so far as the surfacehas any of the properties of a solid, this passive strength is not the same thing as theactive force which the surface could exert to change the shape of the cell. In the secondplace, the greater strength of the concave surface might be a consequence rather than acause of the'folding. The folding, however it is brought about, is bound to involve adiminution in area of the cell surfaces abutting on the-concave side, and this, for instance,by concentrating an ectoplasmic surface layer, might produce an increase in strength ofthese surfaces.

In the absence of any other well-established hypothesis, however, the suggestion thatthe folding of the neural tube is caused by changes of the surface properties of the cellsremains an attractive one. The present investigation has been ooncerned onlywith thecell-water surface. In the embryo itself, forces arising in the cell-cell surfaces would alsohave to be considered. The relations between the forces in the two types of surfacesrequire much further study. Brown et al. (1941), who also tend to attribute neurulationto surface changes, have stressed the possibility that the primary force may be due to'increases (in) the intercellular cohesion or "attraction", resulting in an increased areaof contact between cells' They show that the area of contact does increase. This would


also be expected as a result of effective contraction of the cell-water surfaces on the uppeiside of the neural plate, if this was brought about by an increase in their active surfacetension.. And there is, of course, no reason why cell-water tensions and cell-cell tensionsshould not both be concerned; in fact, one might suppose a priori that both types ofsurfaces would undergo simultaneous, though not necessarily similar, changes.

The discussion has so far tended to minimize the importance of fibrization as a factorin morphogenesis. This may be premature. Although the evidence strongly suggeststhat no importance can be attributed to fibrization within the body of the cytoplasm, thealterations which have just been discussed in the cell surfaces may well depend on thestate of fibrillar aggregation of their protein constituents. Something of the kind, infact, would seem to be the simplest way of accounting for the remarkable polar pro-perties of cells such as those undergoing the invagination movements. Fibrization takingplace in the cell surface might cause an increase in strength; and a similar orientation ofthe fibres in contiguous surfaces might account for the polarity. The positive evidencefor such a suggestion is still meagre, but perhaps some support may be found in theobservations of anisotropy in the thin terminal processes of the flask cells of the blasto-pore (Waddington & Picken, see Waddington, 1940). These thin ends consist mainly ofthe superficial ectoplasmic layer, and the fact that they show double refraction probablyindicates that this layer contains orientated fibrils.

SUMMARY

1. The cells of early amphibian embryos contain ellipsoidal yolk granules. When agranule lies within the mitotic spindle, it is orientated with its long axis parallel to thedirection of the spindle fibres. Yolk granules lying in the cytoplasm of cells are notspecially orientated unless they lie in narrow spaces between cell surfaces; this is truein cells of the moving invagination streams, in the flask cells of the young blastopore, andin elongated cells of the neural groove and lens.

2. The breaking strain of cell surfaces was tested by bringing the cells into surfacesof known strength and observing whether they withstood the applied tension. Theevidence indicates:

(a) That the breaking strain of cells from gastrula and tail-bud embryos is some tensof dynes per centimetre.

(b) That the breaking strain is not the same in all cells, being highest in-neural tissueand lowest in endoderm during the stages mentioned.

(c) That the breaking strain gradually increases with age.(d) That in the neural groove stage the strength of the outer concave surface of the

groove is greater than that of the inner convex surface.3. It is suggested that changes in surface tensions may be of importance in bringing

about morphogenesis. The possibility is envisaged that the changes in tension may beconnected with orientated fibrizations of the surface proteins.


REFERENCES

BROWN, M. G., HAMBURGER, V. & SCHMITT, F. O. (1941). J. Exp. Zool. 88, 353.DARLINGTON, C. D. (1939). The Evolution of Genetic Systems. Cambridge.GADDUM (193a). Proc. Roy. Soc. B, 109, 114.GLASER, O. (1914). Anat. Rec. 8, 525.GLASKR, O. (1916). Science, N.S. 44, 505.HARRISON, R. G. (1936). Coll. Net. 11, 217.HARVEY, E. N. (1937). Trans. Faraday Soc. 33, 943.HARVEY, E. N. & FANKHAUSBR, G. (1933). J. Cell. Comp. Physiol. 3, 463.HOBSON, L. B. (1941). J. Exp. Zool. 88, 107.HOLTFRBTER, J. (1939). Arch. exp. Zellforsch. 23, 169.LANDOLT-BORNSTEIN (1912). Physikalisch-Chemische Tabellen. Berlin.NEEDHAM, J. (1936). Order and Life. Yale.SPEMANN, H. (1931). Arch. EntwMech. Org. 123, 389.WADDINCTON, C. H. (1939a). Introduction to Modern Genetics. London.WADDINGTON, C. H. (19396). Nature, Lond., 144, 637.WADDINOTON, C. H. (1940). Organisers and Genes. Cambridge.WADDINGTON, C. H. (194a). Proc. Zool. Soc. Lond. I l l , 189.

Note added in proof

Since the MS. of this paper was sent to press, I have had an opportunity of seeingthe interesting discussion by F. O. Schmitt {Growth Symposium, 1942) of the importanceof cell surfaces in morphogenesis. Many of the suggestions made there are similar tothose advanced in this paper, and a stimulating discussion is given of the role of proteinaggregates in cell-surfaces.

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